JP4426728B2 - GANP protein - Google Patents

Description

【図面の簡単な説明】
図１は、正常マウスのＰＰにおける２９−１５^＋細胞の検出を示した写真である。２９−１５ｍＡｂ及びＡＬＰ抗ラットＩｇ抗体を用いて、ＰＰについて免疫組織化学的分析を実施した。陽性細胞は、ベクターブルーＡＬＰ基質を有する中央領域に出現し、非特異的内在性ＡＬＰ活性を含む小腸絨毛では、周辺領域に強いシグナルが認められた。二色染色を行うために、切片はビオチン抗Ｂ２２０ｍＡｂ又はビオチン抗ＩｇＤ ｍＡｂのいずれかでさらに染色し、ＤＡＢ及びＨＲＰ−ストレプトアビジンによって発色させた。
図２は、ＳＲＢＣ免疫化マウスのＧＣ領域における２９−１５^＋細胞の出現を示した写真である。正常ＢＡＬＢ／ｃ系マウスは、１２日間に４回ＳＲＢＣを注射し、脾臓切片をヘマトキシリンで染色するか、図１と同様な免疫組織化学染色で調べた。２９−１５ｍＡｂで染色した後に比較すると、正常及びＳＲＢＣ免疫化ＢＡＬＢ／ｃ系マウスの切片は類似していた。
図３は、ＳＲＢＣ免疫化マウスのＧＣ領域における２９−１５^＋細胞の出現を示した写真である。ＧＣ領域の切片を「材料と方法」に記載した個々の色と組み合わせてＰＮＡ、抗ＢｒｄＵ及び２９−１５ｍＡｂで染色した。上段の写真はＧＣ領域（ＧＣ）及び中心溝動脈（ＣＡ）のヘマトキシリン染色を示し、中央の写真は２９−１５^＋ＰＮＡ^＋細胞を指す三色染色を示す。下段は２９−１５^＋ＰＮＡ^＋細胞を図解したものである。
図４は、自己免疫傾向マウスの赤脾髄領域におけるＧＡＮＰ^{ｄｅｎｓｅ＋}細胞の発現を示した写真である。ＢＡＬＢ／ｃ系、ＮＯＤ系、ＮＺＢ系、（ＮＺＢ ｘ ＮＺＷ）Ｆ_１系、ＢＸＳＢ系及びＭＲＬ／ｌｐｒ系の非免疫マウスの脾臓から切片を作製した。全てのマウスは生後６〜８週間で使用した。２９−１５ｍＡｂで染色したＧＡＮＰ^{ｄｅｎｓｅ＋}細胞は、ＮＺＢ系、（ＮＺＢ ｘ ＮＺＷ）Ｆ_１系、ＭＲＬ／ｌｐｒ系及びＢＸＳＢ系の赤脾髄に出現した。
図５は、自己免疫傾向マウスの赤脾髄領域におけるＧＡＮＰ^{ｄｅｎｓｅ＋}細胞の発現を示した写真である。膝窩領域のＬＮ切片を２９−１５ｍＡｂで染色した。ＧＡＮＰ^{ｄｅｎｓｅ＋}細胞は、高齢のＮＺＢ系マウス（１０月齢）及びＭＲＬ／ｌｐｒ系マウス（８週齢）の末梢ＬＮに出現した。
図６は、自己免疫傾向マウスにおけるＧＡＮＰ^{ｄｅｎｓｅ＋}細胞の特徴づけを示した写真である。非免疫ＮＺＢ系マウス（８週齢）の脾臓切片を作製した。下記の試薬の一種と組み合わせて２９−１５ｍＡｂを用いて免疫組織化学的分析を実施した：抗Ｂ２２０、ＰＮＡ。
図７は、自己免疫傾向マウスにおけるＧＡＮＰ^{ｄｅｎｓｅ＋}細胞の特徴づけを示した写真である。非免疫ＮＺＢ系マウス（８週齢）の脾臓切片を作製した。下記の試薬の一種と組み合わせて２９−１５ｍＡｂを用いて免疫組織化学的分析を実施した：抗ＩｇＭ、抗Ｓｙｎｄｅｃａｎ−１、抗ＢｒｄＵ ｍＡｂ。
図８は、ＰＡＳ染色によってＮＺＢ系マウスに現れるモット細胞を示した写真である。
図９は、マウスＧＡＮＰ蛋白質の推定アミノ酸配列を１文字標記で示した図である。
図１０は、ＧＡＮＰ蛋白質の構造を示した図である。図中、Ｓ／Ｔ ｒｉｃｈ ｒｅｇｉｏｎ： セリン／トレオニンが豊富な領域、ＳＡＣ３ ｈｏｍｏｌｏｇｙ ｒｅｇｉｏｎ：ＳＡＣ３相同領域、ｎｕｃｌｅａｒ ｌｏｃａｌｉｚａｔｉｏｎ ｓｉｇｎａｌ：核局在化シグナルを示す。４つのＬＸＸＬＬモチーフが存在する。
図１１は、ｇａｎｐ遺伝子のイン・サイチュＲＮＡハイブリダイゼーションの結果を示した写真である。ＳＲＢＣ免疫、非免疫ＢＡＬＢ／ｃ系、及びＮＺＢ系マウスの脾臓切片をｇａｎｐアンチセンスプローブを用いてハイブリッド形成させた。図中、白脾髄領域（ＷＰ）、赤脾髄領域（ＲＰ）及びＧＣ領域（ＧＣ）を示す。ＮＺＢ系マウスの赤脾髄領域にＧＡＮＰ^{ｄｅｎｓｅ＋}細胞が認められた。
図１２は、ＧＡＮＰ蛋白質を免疫沈降後のウエスタンブロットで分析した結果を示した図である。ＧＡＮＰ蛋白質は、ＷＥＨＩ−２３１系細胞の細胞質分画及び核分画で発現する２１０ｋＤの蛋白質として検出された。
図１３は、正常ＢＡＬＢ／ｃ系マウスの脾臓Ｂ細胞を、ヤギ抗ＩｇＭ抗体（１０μｇ／ｍｌ）及び抗ＣＤ４０ｍＡｂ（１０μｇ／ｍｌ）のＦ（ａｂ’）_２で４８時間刺激し、抗ＧＡＮＰ ｍＡｂで染色した結果を示した図である。
図１４は、抗ＧＡＮＰ免疫沈降物を用いて［γ−^３２Ｐ］−ＡＴＰの存在下で１０分間イン・ビトロのキナーゼ反応を実施した結果を示した図である。蛋白質のリン酸化は、ＳＤＳ−ＰＡＧＥによる分離の後、オートラジオグラフィーによって検出した。ＧＡＮＰのリン酸化を矢印で示し（図Ａ）、リン酸化ＧＡＮＰ蛋白質のホスホアミノ酸分析も示した（図Ｂ）。
図１５は、マウスＧＡＮＰ蛋白質の構造を示した図である。図中、ＳＡＣ３およびＭａｐ８０と相同な領域、核局在化配列（ＮＬＳｓ）、およびコイルド−コイル領域を示す。４つのＬＸＸＬＬモチーフを黒で示す。
図１６は、ＲＴ−ＰＣＲ分析の結果を示す。インビトロでの抗−μ−及び抗−ＣＤ４０−刺激Ｂ細胞におけるｇａｎｐｍＲＮＡの活性化が示されている。ＨＰＲＴをコントロールとして使用し、各鋳型の量を確認した。
図１７は、インビトロキナーゼ反応の結果を示す。細胞溶解物を未刺激（左）または刺激（右）した細胞から調製し、抗ＧＡＮＰ免疫沈降に付した。インビトロキナーゼ反応は［γ−^３２Ｐ］ＡＴＰの存在下において１０分間抗ＧＡＮＰ（４２−２３）免疫沈降物を用いて行った。蛋白質のリン酸化はＳＤＳ−ＰＡＧＥ分離後にオートラジオグラフィーによって検出した。矢印はリン酸化されたＧＡＮＰの位置を示す。
図１８は、ＧＡＮＰとＭＣＭ３の物理的会合を示す図である。ＷＥＨＩ−２３１からの細胞溶解物を抗−ＧＳＴ、抗−ＧＡＮＰ（４２−２３）、または抗−ＭＣＭ３ Ａｂで免疫沈降した。ＳＤＳ−ＰＡＧＥで分離後、蛋白質を電気泳動により膜に移し、抗ＭＣＭ３−Ａｂをプローブとして検出した。
図１９は、ＧＡＮＰとＭＣＭ３の物理的会合を示す図である。ＷＥＨＩ−２３１細胞溶解物からの抗−ＧＳＴ、抗−ＧＡＮＰ（４２−２３）および抗−ＭＣＭ３免疫沈降物をインビトロキナーゼアッセイに付した。正常ウサギ血清（ＮＲＳ）を抗−ＭＣＭ３ Ａｂのためのコントロールとして用いた。試料を７％ＳＤＳ−ＰＡＧＥで分離した。ＧＡＮＰおよびＭＣＭ３に対応するバンドを左側のパネルにおいて矢印で示した。右側のパネルでは、２１０−ｋＤａのバンドのＶ８切断マッピングが同一の切断パターンを示した。コントロールとして、無関係のＶ８消化蛋白質を平行して分離した。
図２０は、抗−ＭＣＭ３ Ａｂと抗−ＣＲ１ｍＡｂまたはＰＮＡとの二重染色を行った結果を示す図である。ＭＣＭ３の発現はＧＣ領域で活性化された。
図２１は、ヒトＧＡＮＰ蛋白質の推定アミノ酸配列を１文字標記で示した図である。
図２２は、ヒト染色体を用いてＦＩＳＨ法によってヒトｇａｎｐとＭａｐ８０をマッピングした結果を示す写真である。Technical field
The present invention relates to a novel protein having kinase activity and a gene encoding the protein.
Background art
Activation and maturation of antigen-specific B cells in peripheral lymphoid tissues is initiated by antigens that bind to membrane IgR (Rajewsky, Nature (Lond.)., 381: 751-758, 1996; Sakaguchi) et al., Adv. Immunol. 54: 337-392, 1993). B cells enter the outside of the periarterial lymph sheath (PALS) (Rajewsky, Nature (Lond.), 381: 751-758, 1996) within 48 hours after immunization and depend on costimulation with specific Th cells. Interaction and dendritic cell finger process association (MacLennan, Annu. Rev. Immunol., 12: 117-139, 1994; Liu et al., Immunol. Rev., 156: 111-126, 1997). ). Antigen-induced B cells proliferate outside of PALS and are then further activated in lymphoid follicles to establish germinal centers (hereinafter sometimes abbreviated as “GC”) (Han). et al., J. Immunol., 155: 556-567, 1995; Jacob et al., J. Exp. Med., 176: 679-687, 1992; Kelsoe, Immunity, 4: 107-111, 1996). These B cells mature and move large during the cell cycle to form dark areas that form large slgs. ⁻ PNA, which becomes a centroblast (centroblast) and is a mature and unique surface feature ⁺ B220 ⁺ slgM ⁺ slgD ⁻ CD38 ⁻ Is expressed in the light region of GC (Kosco-Vilbois et al., Immunol. Today, 18: 225-230, 1997; Kelsoe, Immunol, Today, 16: 324-326, 1995; Oliver et al., J. Immunol., 158: 1108-1115, 1997).
Centrosites are probably undergoing either apoptosis or affinity maturation of the immunoglobulin V region and the process of changing the class switch to IgG class antigens, but some centrosites are lymphoid as memory B cells Survives for a long time in the fraction. Other centrosites probably migrate to the peripheral zone of GC and receive further antigenic stimulation and costimulatory signals via receptors for B cell activating molecules such as CD40 and CD38 and various B cell stimulating cytokines (Gray et al., J. Exp. Med., 180: 141-155, 1994; Foy et al., J. Exp. Med., 180: 157-163, 1994). Antigen-specific B cells that are further stimulated in this region are probably interstitial regions of the spleen (called the red pulp), where various other immunocompetent cells may interact with antigen-derived B cells. Run in. Histochemical analysis of several autoimmune mice has identified unique antibody-producing cells in this region that appear as plasma cells or as abnormal types of plasma cells called Mott cells (Tallinton et al. Eur. J. Immunol., 22: 531-539, 1992; Jiang et al., J. Immunol., 158: 992-997, 1997).
Autoimmunity is a phenomenon in which self / non-self discrimination impairment occurs frequently in antigen-specific lymphocytes (Theofilopoulos, Immunol. Today, 16: 90-98, 1995). In the immune system of various autoimmune diseases, a complex mechanism involving T cells and B cells is observed (Theofilopoulos et al., Adv Immunol., 37: 269-290, 1985; Okamoto et al., J. MoI. Exp.Med., 175: 71-79, 1992; Reininger et al., J. Exp.Med., 184: 853-861, 1996; Theofilopoulos et al., Immunol. Rev., 55: 179-216, 1981. Watanabe-Fukunaga et al., Nature (Lond.)., 356: 314-317, 1992; Takahashi et al., Cell, 76: 969-976, 1994; . T al, Nature, 328 (Lond.):. 805-811,1987).
The NZB and NZW systems are (NZB x NZW) F ₁ As a strain mouse, it has been characterized by a number of genetic factors that cause SLE, a severe autoimmune condition (Theofilopoulos et al., Adv. Immunol., 37: 269-290, 1985; Okamoto et al.,). J. Exp. Med., 175: 71-79, 1992; Reininger et al., J. Exp.Med., 184: 853-861, 1996; Theofilopoulos et al., Immunol. Rev., 55: 179-216. , 1981). In NZB mice, an autoimmune state is spontaneously formed by anti-erythrocyte antibodies that cause autoimmune hemolytic anemia (Okamoto et al., J. Exp. Med., 175: 71-79, 1992), NZW mice exhibit an insidious autoimmune phenomenon (Reininger et al., J. Exp. Med., 184: 853-861, 1996). (NZB x NZW) F ₁ The SLE status of strain mice is clearly caused by a number of genetic factors associated with T and B cells (Theofilopoulos et al., Immunol. Rev., 55: 179-216, 1981). NZB mice show obvious abnormalities of B cells, but the molecular mechanism of abnormal B cell activation in NZB mice has not been elucidated.
Disclosure of the invention
In order to examine such molecular problems related to B cell maturation, the present inventors produced monoclonal antibodies against intracellular components of the WEHI-231 system, which is a mouse B cell line. This cell line has a NZB line genetic background, and the monoclonal antibody designated 29-15 recognizes a differentiation antigen whose expression is enhanced in GC-B cells of peripheral lymphoid tissues. Using this 29-15 monoclonal antibody, the present inventors examined the expression of an antigen in peripheral lymphoid tissues, and this antigen was characterized as a differentiation antigen enhanced in the bright region of GC in hyperimmune mice. . In the spleen of NZB mice, IgM-producing plasma cells that highly express GANP antigen appear before the start of autoimmunity. This expression is an important molecular behavior for understanding peripheral immune responses and autoimmunity by autoantibodies. It was suggested that there is.
The present inventors have conducted research to confirm the above-mentioned antigen whose expression is selectively enhanced in the centrosite at the germinal center, and in situ RNA using an isolated cDNA probe (ganp probe). It was confirmed by hybridization that the expression of ganp mRNA was increased in the region stained with the 29-15 monoclonal antibody. It was also confirmed that the gene product GANP protein is a 210 kD protein localized in the cytoplasm and nucleus and structurally similar to the yeast transcriptional regulatory gene SAC3. When B cells were activated with anti-IgM antibody and anti-CD40 antibody, the amount of kinase bound to GANP protein increased. From these results, it was suggested that GANP protein may be involved in signal conversion of abnormal B cell differentiation in a certain autoimmune state. The present invention has been completed based on these findings.
That is, the present invention provides a GANP protein specified by the amino acid sequence set forth in SEQ ID NO: 1 or 3 in the sequence listing. According to the present invention, in the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 in the sequence listing, one or more amino acids are deleted, one or more amino acids are replaced with other amino acids, and / or one or more A GANP mutant protein having an amino acid sequence to which other amino acids are added and having the same kinase activity as that of the GANP protein is provided. According to the present invention, a polypeptide comprising the full-length amino acid sequence of the GANP protein or GANP mutant protein as a partial sequence thereof is provided.
From another aspect, the present invention provides a polynucleotide encoding the above-mentioned GANP protein or GANP mutant protein. A typical polynucleotide is DNA encoding a GANP protein derived from a mammal, and among them, a gene DNA possessed by a mammal is preferable. The most preferable example of the polynucleotide is specified by the nucleotide sequence described in SEQ ID NO: 2 (DNA sequence encoding mouse-derived GANP protein) or SEQ ID NO: 4 (DNA sequence encoding human-derived GANP protein). Is done.
The present invention also provides an antisense polynucleotide comprising the nucleotide sequence of the antisense strand of the polynucleotide or a derivative of the antisense polynucleotide. Furthermore, according to the present invention, a polynucleotide or an antisense polynucleotide which is a part of the polynucleotide or the antisense polynucleotide and which comprises 12 or more consecutive bases, and the polynucleotide or the antisense Polynucleotides or antisense polynucleotides that are chemically modified polynucleotides are provided.
From another point of view, according to the present invention, there is provided a method for obtaining DNA comprising the nucleotide sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4 in the sequence listing or DNA homolog derived from other mammals, A method for obtaining cDNA hybridizing with a probe from a cDNA library of mammals using a nucleotide or an antisense polynucleotide as a probe is provided. The length of the cDNA is almost the same as that of the GANP gene, and the protein encoded by it is about 210 kDa. The present invention also provides the cDNA obtained by the above method and the GANP protein encoded by the cDNA.
From another aspect of the present invention, an antibody that recognizes GANP protein or GANP mutant protein is provided.
Preferred form for carrying out the invention
A representative example of the GANP protein of the present invention is a protein specified by the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 in the sequence listing, characterized by having a molecular weight of 210 kDa and having kinase activity. The GANP mutant protein provided by the present invention has 1 to several, preferably 1 to 20, more preferably 1 to 10, and most preferably 1 amino acid sequences described in SEQ ID NO: 1 or SEQ ID NO: 3. About ~ 5 amino acid residues are specified by the substituted, inserted, and / or deleted amino acid sequence, and are substantially similar to the GANP protein specified by the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3. Has kinase activity. Any of these GANP mutant proteins are included in the scope of the present invention. The protein specified by the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 and its homologue in the sequence listing are proteins whose expression is selectively enhanced in the centrosite at the germinal center of the mammal from which the protein is derived.
Usually, the active domain of GANP protein or GANP mutant protein is a polypeptide in which amino acid residues are deleted from the N-terminal and / or C-terminal of the full-length amino acid sequence, and the kinase activity of the polypeptide is measured. This can be easily confirmed. The polypeptide provided by the present invention is a polypeptide comprising the active domain of GANP protein and GANP mutant protein, and a polypeptide comprising a polypeptide comprising the active domain as a partial sequence, which is substantially the same as GANP protein. Has kinase activity. Another polypeptide provided by the present invention is a polypeptide containing the full-length amino acid sequence of GANP protein or GANP mutant protein as a partial sequence thereof, and has substantially the same kinase activity as GANP protein.
The polynucleotide provided by the present invention encompasses all polynucleotides obtained by chemically modifying DNA or RNA in addition to DNA and RNA. As used herein, the term “polynucleotide” should be construed in its broadest sense, including non-naturally occurring. A representative example of the polynucleotide provided by the present invention is DNA or RNA encoding the above-mentioned GANP protein or GANP mutant protein. Another example of the polynucleotide of the present invention is an antisense polynucleotide.
By utilizing the degeneracy of the genetic code, at least a part of the base sequence of the polynucleotide can be replaced with another type of base without changing the amino acid sequence of the polypeptide produced from the polynucleotide. Are well known to those skilled in the art. Therefore, the polynucleotide of the present invention includes all polynucleotides encoding GANP protein or GANP mutant protein. As an example of a preferable gene of the present invention, a gene encoding a mouse-derived GANP protein is shown in SEQ ID NO: 2 in the sequence listing, and a gene encoding a human-derived GANP protein is shown in SEQ ID NO: 4. The amino acid sequence of the GANP mutant protein can be determined from the base sequence of the gene encoding the mutant. For example, it can be performed using a commercially available program (for example, MacVector (registered trademark, manufactured by Eastman Chemical Co.) or Genetix (Software Development).
The scope of the present invention includes an antisense polynucleotide comprising a base sequence of an antisense strand of a polynucleotide encoding a GANP protein and derivatives thereof. An antisense polynucleotide is provided as an embodiment of the above-described polynucleotide. In the present specification, there is a case where it is referred to as an “antisense polynucleotide” particularly when it is clearly indicated that the polynucleotide is composed of a base sequence of an antisense strand. is there. The antisense polynucleotide is capable of hybridizing to a polynucleotide encoding GANP protein, and if the polynucleotide to be hybridized is a polynucleotide of the coding region, the polynucleotide of the polypeptide encoded by the polynucleotide It is possible to inhibit biosynthesis.
The antisense polynucleotide for inhibiting polypeptide biosynthesis preferably comprises 12 bases or more. On the other hand, a sequence longer than necessary is not preferable in order to incorporate a full-length antisense polynucleotide into a cell. In the case of incorporating an antisense polynucleotide into a cell and inhibiting the biosynthesis of GANP protein, the base is 12 to 30 bases, preferably 15 to 25 bases, more preferably 18 to 22 bases. An antisense polynucleotide consisting of
The antisense polynucleotide or derivative thereof of the present invention includes all of a plurality of nucleotides composed of a base, a phosphate, and a sugar, regardless of whether or not they exist in nature. Typical examples are natural antisense DNA and antisense RNA. Examples of non-naturally occurring polynucleotides include methylphosphonate and phosphorothioate polynucleotides. The antisense polynucleotide of the present invention is excellent in binding ability to target DNA or mRNA, tissue selectivity, cell permeability, nuclease resistance, intracellular stability, etc. using antisense technology available to those skilled in the art. A variety of antisense polynucleotide derivatives are obtained.
In general, in terms of ease of hybridization, it is preferable to design an antisense polynucleotide or a derivative thereof having a base sequence complementary to the base sequence of a region forming an RNA loop. Therefore, the antisense polynucleotide of the present invention and its derivative are also preferable examples that hybridize to the loop region of RNA. In addition, an antisense polynucleotide having a sequence complementary to the sequence of a translation initiation codon, a ribosome binding site, a capping site, or a splice site can generally be expected to have a high expression suppression effect. Therefore, the antisense polynucleotide of the present invention or a derivative thereof, comprising a sequence near the translation initiation codon of a gene encoding GANP protein, a complementary sequence of a ribosome binding site, a capping site, and / or a splice site, This is a preferred embodiment from the viewpoint of expression suppression effect.
Among the currently known polynucleotide derivatives, those having at least one of nuclease resistance, tissue selectivity, cell permeability, and binding force, preferably having a phosphorothioate bond as a skeleton structure Derivatives can be mentioned. The polynucleotide and derivatives thereof of the present invention include derivatives having these functions or structures.
Of the antisense polynucleotides of the present invention, natural antisense polynucleotides can be synthesized using a chemical synthesizer, or can be produced by PCR using DNA encoding GANP protein as a template. In addition, polynucleotide derivatives such as methyl phosphonate type and phosphorothioate type can be usually produced by chemical synthesis. In this case, the operation can be performed according to the instructions attached to the chemical synthesizer, and the resultant synthesis product can be purified by HPLC using reverse phase chromatography or the like.
A polynucleotide encoding the GANP protein of the present invention, an antisense polynucleotide thereof, or a polynucleotide that is a part thereof (for example, a polynucleotide comprising 12 or more consecutive bases) is obtained from a mammalian cDNA library using a GANP protein. It can be used as a probe for screening DNA encoding. For such purposes, a polynucleotide comprising a base sequence of 15 or more consecutive bases is particularly preferred. The polynucleotide used as a probe may be a derivative. Usually, a sequence having the number of bases or more is recognized as a specific sequence.
A DNA consisting of 12 or more consecutive bases of the base sequence set forth in SEQ ID NO: 2 or 4 in the sequence listing or a polynucleotide hybridizing to the DNA (antisense polynucleotide) is a DNA encoding a GANP protein Can be used as a probe for screening from a cDNA library or the like.
Furthermore, by performing Northern blot hybridization on mRNA derived from each tissue using a polynucleotide encoding the GANP protein of the present invention or an antisense polynucleotide thereof, or a polynucleotide which is a part thereof as a probe, the gene is derived from the GANP gene. It is possible to find a tissue in which the mRNA is expressed. Furthermore, a polynucleotide comprising 12 or more bases can be used as a primer for polymerase chain reaction (PCR), and a polynucleotide encoding a GANP protein can be obtained by PCR. In addition, any part of the GANP protein can be cloned by appropriately selecting primers.
As a cDNA library used in the screening using the probe, those prepared from mRNA can be preferably used. A group of cDNAs selected by random sampling from these cDNA libraries can be used as search samples. A commercially available cDNA library can also be used.
A transformant can be prepared by inserting a cDNA hybridizing with the GANP gene obtained above into an appropriate vector (eg, pGEX-4T-1 vector) and then introducing it into a host (eg, E. coli). . The type of vector and the type of host are not particularly limited, but an appropriate expression vector can be selected and used according to the type of host. As the host, bacteria such as Escherichia coli, yeast, or animal cells can be used. The method for obtaining a transformant by introducing a recombinant vector into an appropriate host such as Escherichia coli is not particularly limited, and any method available to those skilled in the art is applicable.
It is possible to produce a GANP protein by culturing a transformant introduced with the GANP gene of the present invention and amplifying the gene DNA or expressing the protein. There are various books and reports on the production and culture of transformants, and various means have been developed and widely used in the industry. Therefore, those skilled in the art can easily produce a GANP protein based on the base sequence described in the present specification. As a technique for introducing a gene into a cell, a calcium chloride method, a lipofection method, a protoplast method, an electroporation method, or the like can be used.
Separation and purification of the target protein from the culture can be performed by appropriately combining means available to those skilled in the art. For example, the GANP protein of the present invention can be efficiently recovered and purified by performing operations such as concentration, solubilization, dialysis, and various chromatography as required. More specifically, immunoprecipitation, salting-out, ultrafiltration, isoelectric precipitation, gel filtration, electrophoresis, ion exchange chromatography, hydrophobic chromatography, antibody chromatography, etc. The various affinity chromatography, chromatofocusing method, adsorption chromatography method, reverse phase chromatography method and the like may be appropriately selected. By using a gene encoding a GANP mutant protein, a GANP mutant protein can be similarly produced.
The GANP protein or GANP mutant protein can also be produced as a fusion peptide with another polypeptide. Such fusion peptides are also encompassed within the scope of the present invention. The type of polypeptide to be fused is not particularly limited, and examples thereof include a signal peptide that promotes extracellular secretion. Such a fusion protein can also be produced using a transformant. When a GANP protein or a GANP mutant protein is produced using a fusion protein, the fusion protein may be treated with a chemical substance such as bromocyan or an enzyme such as protease, and the cut out target product may be separated and purified.
Using the GANP protein or GANP mutant protein of the present invention, or a partial polypeptide thereof, an antibody that recognizes the GANP protein or GANP mutant protein can be produced. The antibody of the present invention can be produced according to a method commonly used in the art by immunizing a mammal with a GANP protein or a GANP mutant protein. It can be confirmed that the antibody recognizes the GANP protein or GANP mutant protein of the present invention by Western blotting, ELISA, immunostaining (for example, measurement by FACS) or the like. As an immunogen, in addition to GANP protein or GANP mutant protein, those obtained by binding a part thereof to other carrier protein such as bovine serum albumin may be used. A part of GANP protein or GANP mutant protein preferably has 8 amino acid residues or more, and such a polypeptide may be synthesized using, for example, a peptide synthesizer.
In addition, a monoclonal antibody produced from a hybridoma produced using lymphocytes of an immunized animal may be used as the antibody of the present invention. Monoclonal antibody production methods are well known in the art and widely used ("Antibodies, A Laboratory Manual" (Cold Spring Harbor Laboratory Press, 1988), Chapter 6). As the antibody of the present invention, it is also possible to use an antibody fragment or a chimeric antibody having antigen-antibody reaction activity. The GANP protein or GANP mutant protein of the present invention can be detected by a method using an antibody or a method using an antibody and an enzyme.
Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited to the following examples.
Example
Example 1: Cloning and expression analysis of mouse GANP gene
<Materials and methods>
(1) Animals and immunization
BALB / c mice and Lewis rats were purchased from Seac Yoshitomi Ltd. (Fukuoka). NZB, NZW, (NZB x NZW) F ₁ Strain mice (7 weeks old, female), MRL / lpr mice (8 weeks old, female) and BXSB mice (7 weeks old, male) were obtained from Japan SLC (Shizuoka). Elderly NZB mice (10 months old, female) were donated by Dr. Sachiko Hirose (Juntendo University School of Medicine, Department of Pathology). NOD mice (7 weeks old, male) were provided by Dr. Junichi Miyazaki (Osaka University School of Medicine, Department of Nutrition and Physiology). All animals were bred at the Animal Research and Development Center at Kumamoto University. BALB / c mice were immunized multiple times with sheep erythrocytes (Japan Biotest Laboratories, Tokyo). Immunization was performed intravenously at 5-day intervals, and thymus, spleen, lymph node (LN) and Bayer's plate (PP) sections were prepared for immunohistochemical analysis.
(2) Cells and cell culture
Spleen B cells of BALB / c mice were grown as previously reported (Nomura et al., Immunol. Lett., 45: 195-203, 1995). The cells were inactivated in an incubator containing 5% carbon dioxide at 37 ° C., heat-inactivated FCS (Dainippon Pharmaceutical Co., Ltd., Osaka) 10%, L-glutamine (Biowhitteker, Walkersville, MD, USA) 5 mM, penicillin 100 U / ml And RPMI-1640 medium (Gibco-BRL, Gaithersburg, Germany) containing streptomycin 100 μg / ml and 2-ME 50 μM.
(3) Establishment of 29-15 monoclonal antibody (hereinafter abbreviated as “29-15 mAb”)
(BALB / c x NZB) F ₁ A mAb against the WEHI-231 strain, which is a mouse B cell line established using mineral oil, was prepared by the method described previously (Kuwahara et al., J. Immunol., 152: 2742-22752, 1994). . Briefly, the surface phenotype slgM was determined by the method of Sakaguchi et al. (Sakaguchi et al., EMBO (Eur. Mol. Biol. Organ.) J., 5: 2139-2147, 1986). ⁺ slgD ⁺ B220 ⁺ A WEHI-231 cell lysate having was prepared in hypotonic buffer in the absence of detergent and dialyzed against phosphate buffer (PBS). This cell lysate was suspended in complete Freund's adjuvant (CFA) (Difco Laboratories, Detroit, Michigan, USA) and administered into the footpad of Lewis rats to immunize the mice, and incomplete Freund's adjuvant (IFA) (Difco Laboratories), and booster immunization was performed twice on the 4th and 8th days. Nine days later, lymph nodes in the popliteal and inguinal regions were excised, and a lymphoid cell suspension was prepared. Establishment of hybridoma, selection of HAT medium (Gibco-BRL), and recloning of hybridoma clones were performed as previously described (Kuwahara et al., J. Immunol., 152: 2742-22752, 1994). 29-15 mAb that stains lymphoid cells in immunohistochemical analysis was selected.
(4) Antibodies and reagents
F (ab ′) of affinity purified goat anti-mouse μ antibody ₂ Fragments (ICN Pharmaceutical, Inc., Costa Mesa, CA, USA), biotin-conjugated peanut agglutinin (PNA) (Vector Laboratories, Inc., Burlingame, CA, USA), biotin-conjugated anti-CD35 mAb (PharMingen, San Diego, CA) Alkaline phosphatase (ALP) -conjugated goat anti-rat IgAb (# 59301, ICN), HRP-conjugated goat anti-rat IgAb (ICN), HRP-conjugated streptavidin (Kirkegaard & Perry Laboratories, Inc., Gaitherburg, MD), ALP-conjugated Mouse IgAb (Sigma Chemicals Co., St. Louis, MI), FITC Mouse anti-rat κmAb (ICN), PE conjugated anti -B220mAb (PhaMingen), and ALP-conjugated goat anti-rabbit IgAb (Zymed Laboratories Inc., South San Francisco, CA) were used as purchased. Biotin-conjugated mAbs such as anti-B220 (RA3-6B2), anti-μ (AM / 3) and anti-δ (CS / 15) were made in our laboratory. Anti-CD40 mAb (LB429) was established in our laboratory (Nomura et al., Immunol. Lett., 45: 195-203, 1995). AM / 3 and CS / 15 hybridomas were provided by Dr. Kensuke Miyake (Saga Medical University, Department of Immunology). Biotin-conjugated anti-Syndecan-1 was purchased from PharMingen (San Diego, CA, USA). Anti-BrdUmAb was obtained from Novocastra Laboratories, Ltd. (Newcastle, UK). Rabbit anti-mouse MCM3 / P1 Ab has been described in the literature (Kimura, H et al., 1994, EMBO J. 13, 4311-4320).
(5) Immunohistochemistry
Immunohistochemical staining was performed as previously reported (Ezaki et al., Arch. Histol. Cytol., 58: 104-115, 1995; Yamanouchi et al., Eur. J. Immunol., 28: 696-707). 1998). In brief, BALB / c, NZB, (NZB x NZW) F ₁ Target organs excised from strains, NOD strains, BXSB strains and MRL / lpr mice were placed in OCT compounds (Miles Inc., Elkhart, Indiana, USA). A 6 μm frozen section placed on a gelatin-coated slide was completely air-dried. The slide glass was then fixed with acetone for 10 minutes, followed by rehydration with PBS for 15 minutes. Slides were incubated with 29-15 mAb for 60 minutes and washed several times with PBS. After incubation with alkaline phosphatase-conjugated goat anti-rat Ig antibody (ALP-anti-rat Ig, catalog number 59301, ICN Pharmaceutical, Inc.), the slides were washed 4 times with PBS. The slides were developed using Vector Blue (Vector Laboratories).
For secondary staining, slides were incubated with biotin-labeled mAb and horseradish peroxidase (HRP) -conjugated streptavidin (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD, USA). After developing with p-dimethylaminoazobenzene (DAB, Dojindo Laboratories, Kumamoto), the sections were lightly fixed with 1% glutaraldehyde / PBS solution. In order to detect actively proliferating cells in vivo, BrdU (Sigma Chemicals Co., St. Louis, Mo., USA) was injected intravenously 1 hour before organ collection. Cells undergoing DNA synthesis were detected by staining with anti-BrdU mAb and ALP-conjugated goat anti-mouse Ig antibody (Sigma Chemicals Co.) and developed with Vector Red (Matsuno et al., Cell Tissue Res. 257: 459-470, 1989). Periodic acid Schiff (PAS) staining was performed as previously reported (Jiang et al, J. Immunol., 158: 992-997, 1997). All sections were mounted with Aquatex (E. Merck, Darmstadt, Germany).
(6) Molecular cloning of cDNA using λgt11 vector
After transferring the fusion protein onto a nitrocellulose filter (Schleicher and Schuell, Darmstadt, Germany) presoaked in 20 mM IPTG, the supernatant of 29-15 mAb was used to obtain mouse spleen, mouse bone marrow, WEHI-231 cells and A20. A cDNA library constructed with mRNA from cells was screened (Inui et al., J. Immunol., 154: 2714-2723, 1995). The phage plate was incubated at 42 ° C. for 4 hours, then the plate was covered with a filter and further incubated at 37 ° C. for 4 hours. Filters were washed 3 times with wash buffer (PBS containing Tween 20 0.1%), blocked in block buffer (PBS containing 0.1% Tween 20, 5% non-fat dry milk) for 1 hour, and then 29-15 mAb Incubated with. ¹²⁵ Positive signals were detected by autoradiography using I-labeled sheep anti-rat Ig antibody (Amersham, Buckinghamshire, UK). The first cDNA clone contained a 280 bp fragment capable of encoding the polypeptide as a fusion protein. Using this first 280 bp fragment, a longer cDNA clone was isolated from another WEHI-231 cDNA library. The 4.9 kb fragment of the secondary cDNA clone encoded the longest 4.5 kb open reading frame. In order to further determine the 5 ′ sequence, a race kit from Gibco-BRL using the 5′-RACE method was used.
(7) In situ RNA hybridization on tissue sections
In situ RNA hybridization was performed as previously reported (Kondo et al., Blood, 80: 2044-2051, 1992). Paraffin-embedded sections were mounted on silanized glass slides. After deparaffinization of the slide glass, hybridization was carried out at 50 ° C. for 16 hours using a ganp 280 bp riboprobe labeled with digoxigenin. The slide glass was washed several times with TNE buffer (10 mM Tris-HCl [pH 7.6], 500 mM sodium chloride, 1 mM EDTA) at 37 ° C., and then washed with 2 × and / or 0.2 × SSC solution at 50 ° C. did. Color was developed in the presence of ALP substrate using an anti-digoxigenin antibody.
(8) Preparation of GST-cDNA fusion protein and another anti-GANP mAb
A ganp cDNA fragment encoding a part of GANP (amino acids 679 to 1028 of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing) was introduced into the pGEX-4T-1 vector (Pharmacia Biotech, Piscataway, NJ, USA). . Recombinant plasmids were confirmed by DNA sequencing for all insertions and linkages. A GST-GANP fusion protein was prepared by glutathione-Sepharose (chromia) column chromatography as previously reported (Inui et al., J. Immunol., 154: 2714-2723, 1995). An anti-GANP mAb, designated 42-43, was established by immunization of the fusion protein in rats as described above.
(9) Western blot analysis
Protein gel electrophoresis, Western blot transfer and protein immunodetection were performed as previously reported (Kuwahara et al., Int. Immunol., 8: 1273-1285, 1996). 50 million cells were treated with 1 ml of TNE lysis buffer (10 mM Tris-HCl [pH 7.8], 150 mM sodium chloride, 1 mM EDTA, 1% NP-40, 0.02% NaN. ₃ ) And the immune complex was analyzed by SDS-PAGE (7%). After transferring the protein onto a nitrocellulose filter, the filter was blocked with PBS-Tween 20 containing 5% non-fat dry milk and incubated with anti-GANP mAb for 60 minutes. After several washes with PBS-Tween 20, the filters were incubated with HRP-conjugated goat anti-rat Ig (ICN Pharmaceutical, Inc.) for 30 minutes. Color was developed using an ECL detection kit (Amersham).
(10) Subcellular fractionation
Complete nuclei were isolated as previously reported (Schriber et al., Nucleic Acids Res., 17: 6419, 1989). WEHI-231 cells were washed with TBS, and the pellet was re-incorporated in buffer A (10 mM HEPES [pH 7.9], 10 mM potassium chloride, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF). Suspended, incubated on ice for 15 minutes, and finally added NP-40 to 1%. After centrifugation, the supernatant was collected as a cytoplasmic fraction. The pellet was resuspended and homogenized with the same buffer, and complete nuclei were obtained by staining. The sample was centrifuged and the pellet was resuspended in cold buffer C (20 mM HEPES [pH 7.9], 0.4 M sodium chloride, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF) and centrifuged. The supernatant was frozen as a nuclear fraction at -80 ° C.
(11) In vitro kinase reaction and phosphoamino acid analysis
In vitro kinase reaction was performed using immunoprecipitates as previously described (Kuwahara et al., J. Immunol., 152: 2742-22752, 1994). Spleen B cells were purified by the method described previously (Nomura et al., Immunol. Lett., 45: 195-203, 1995). B cells have been reported in vitro (Nomura et al., Immunol. Lett., 45: 195-203, 1995) in vitro with goat anti-IgM antibody and anti-CD40 mAb (LB429) F (ab '). ₂ The fractions were stimulated for 48 hours. Cells were collected and washed, then lysed with TNE lysis buffer and immunoprecipitated with anti-GANP mAb (42-23). The immunoprecipitate is [γ- ³² After incubation with P] -ATP (Amersham), radiolabeled proteins were analyzed by autoradiography using SDS-PAGE (7%). The band corresponding to GANP was removed from the dried gel. After SDS was removed from the gel, the homogenized gel was digested overnight at 37 ° C. with TPCK-trypsin (Sigma Chemicals Co.). Samples were hydrolyzed with 6N hydrochloric acid and electrophoresed on TLC (E. Merck).
V8 cleavage mapping of the indicated proteins was performed as previously reported (Kuwahara, K., et al., 1994, J. Immunol. 152: 2742-2275).
(12) Cytoplasmic staining
Cells were fixed with 2.5% paraformaldehyde / PBS solution and permeabilized with 70% ethanol on ice for 1 hour. Cells were incubated with 29-15 mAb and FITC-conjugated mouse anti-rat κ mAb. Antibody binding was analyzed by FACScan flow cytometry (Becton-Dickinson, Mountain View, CA, USA).
(13) Immunoprecipitation-Western blot analysis
The protein obtained by (10) subcellular fractionation was immunoprecipitated with anti-GANP mAb in combination with protein G-Sepharose and analyzed by SDS-PAGE. Western blot filters were incubated with anti-GANP mAb and then with HRP-anti-rat Ig. Development was performed using an ECL detection kit (Amersham).
(14) Reverse transcriptase PCR (RT-PCR)
Total RNA (1 μg each) purified from cultured B cells using TRISOL (Gibco BRL, Rockville, MD) was used as a template for cDNA synthesis (100 μl volume) using Superscript (Gibco BRL). PCR amplification was performed using Taq-Gold (Perkin-Elmer, Foster, Calif.) And primers for ganp or HPRT (control) (Han, S., et al., 1996. Science. 274: 2094-2097) This was performed using 2 μl of each cDNA solution. The ganp transcript was amplified by 5′-CCGTGGGATGACCATCATCAC-3 ′ (forward primer) (SEQ ID NO: 5 in the sequence listing) and 5′-CATGTCCACCATCTCCAGCA-3 ′ (reverse primer) (SEQ ID NO: 6 in the sequence listing).
<Result>
(1) Expression of GANP antigen in lymphoid organs
A mAb that recognizes a differentiation antigen expressed in peripheral B cells was prepared by immunizing rats with a lysate of WEHI-231 cells. For normal lymphoid organs in BALB / c mice, immunohistochemical analysis using 29-15 mAb did not detect expression in the bone marrow, but was less in lymphoid organs such as thymus, spleen and lymph nodes Expression was observed. A small number of cells strongly expressed the 29-15 antigen in the splenic red pulp and lymph node subcortex. It is interesting that the expression was very strong in the central region of PP follicles (FIG. 1). Cells are positive with anti-B220 mAb, while not positive with anti-IgD mAb. Normal mice received secondary lymphoid follicles with distinct GC in PP for continuous stimulation of various antigenic substances introduced through the small intestinal lumen.
By repeating immunization with sheep red blood cells (SRBC), lymphoid follicles were formed in the spleen within 12 days. By antigen immunization, 29-15 in the GC region of the spleen and lymph nodes in addition to the GC of PP ⁺ Cells appeared (Figure 2). The 29-15 antigen was enhanced in GC cells. 29-15 in the structure of secondary lymphoid follicles ⁺ Further analysis of the cell phenotype revealed that almost half of the PNA ⁺ GC-B cells were positive for 29-15 mAb while negative for anti-BrdU mAb (FIG. 3). Interestingly, expression of the 29-15 antigen was enhanced in centrosite at the distal region of the entrance from the central groove artery. This phenotype is consistent with the GC-B cell criteria and, as already mentioned, supports the name “GANP” of the 29-15 antigen.
(2) Autoimmunity tendency GANP in the red pulp region of NZB mice ^{dense +} Appearance of B cells
In the absence of in vivo stimulation, normal mice are almost GANP in the follicular area of the spleen ⁺ GANP does not express B cells, but GANP protein is remarkably expressed in the red pulp region of BALB / c system (FIG. 2) and C57BL / 6 system ^{dense +} Some cells were observed. These cells are large and clearly different from normal B cells. However, in young NZB mice (8 weeks old), this GANP ^{dense +} Cells spontaneously increased in the red pulp region of the spleen without immunity (FIG. 4). In another autoimmune-prone mouse, the NZW strain, GANP in the red pulp at 5-12 weeks of age ^{dense +} Cells were not expressed but are a serious disease combination (NZB x NZW) F ₁ In the system, GANP in the red pulp ^{dense +} Cells were moderately expressed.
GANP in the spleen of other autoimmune-prone mice ^{dense +} We examined whether cells also appear naturally. GANP ^{dense +} The cells appeared in the BXSB and MRL / lpr spleens, but no significant appearance was observed in similarly-week-old NZW and NOD mice without specific pathogens (SPF). GANP ^{dense +} Cells became distinguishable during aging and appeared in peripheral lymph nodes of diseased aged NZB mice (10 months old). GANP ^{dense +} Most of the cells appear in the late stage in the lymph node, and in the MRL / lpr system, the GANP enters the lymph node in the young stage (8 weeks old). ^{dense +} The appearance of cells is particularly interesting (Figure 5). From these results, the genetic factors of NZB, BXSB and MRL / lpr mice with autoimmunity tend to be GANP in the red pulp region. ^{dense +} It is suggested that it may control the appearance of cells and recruitment into lymph nodes.
GANP in the red pulp by two-color analysis ^{dense +} Cell phenotype is PNA ⁻ B220 ⁻ Cells (Figure 6) and IgD ⁻ CD38 ⁻ Shown as a cell. These cells are positive when stained with anti-Syndecan-1 mAb that selectively stains plasma cells. GANP ^{dense +} Although the cells express IgM in the cytoplasm (FIG. 7), it is possible that these cells are Mott cells (Jiang, Y., S. Hirose, Y. Hamano, S. Kodera, H. Tsurui, M). Abe, K. Terashima, S. Ishikawa and T. Shirai. 1997. J. Immunol. 158: 992-997), the sections were stained with PAS staining, and GANP ^{dense +} The cell is B220 ⁻ Syndecan-1 ⁺ PNA ⁻ BrdU ⁻ GANP ^{dense +} (Figure 8) and CD40 ⁻ CD38 ⁻ With PAS ⁻ Met. These plasma-like cells appear preferentially in the spleen of NZB mice, but are different from the currently reported Mott cells.
(3) Identification of cDNA clone encoding GANP antigen
We used 29-15 mAb to isolate a candidate cDNA clone (with 280 bp insert DNA) from a WEHI-231 strain cDNA library and a longer cDNA named ganp. By determining the full length of the nucleotide sequence (6429 bp) with overlapping clones, a polypeptide consisting of 1971 amino acids with a predicted molecular size of 210 kD was estimated (FIG. 9). The amino acid sequence of GANP protein is shown in SEQ ID NO: 1 in the sequence listing, and the base sequence of ganp cDNA is shown in SEQ ID NO: 2 in the sequence listing.
The GANP amino acid sequence is SAC3 and human Map80 protein (Takei, Y et al., 1998, J. Biol. Chem.), Which is thought to be a nuclear transcriptional regulator characteristic of the temperature mutant Saccharomyces cerevisiae. 273: 22177-22180) (FIGS. 10 and 15; Bauer, A. and R. Koelling. 1996. Yeast 12: 965-975). GANP protein also exhibits low homology within short regions of various transcription factors including insulin promoting factors (amino acids 996 to 1063) and NF-IL-6 (amino acids 388 to 450).
The GANP gene has a consensus base sequence of a superhelical motif, but no zinc finger, leucine zipper and homeodomain motifs are observed. A serine / threonine-rich region was found in the N-terminal 100 amino acids, but this region was slightly homologous to the nucleoporin known as the nuclear pore complex. GANP consists of two nuclear localization sequences ( ⁴⁹⁷ HKKKK and ¹³⁴⁴ PMKQKRR), but these may be involved in the expression of GANP in the nucleus, as suggested by the PSORT program. GANP also has two coiled-coil motifs, but no zinc fingers, leucine zippers or homeodomain motifs. In addition, there were four LXXLL motifs found in nuclear transcription co-activator molecules including CBP / p300 and p / CIP (Torcia J. et al., 1997, Nature (Lond.), 387. : 677-684; Heery et al., 1997, Nature (Lond.), 387: 733-736), no associated molecules have been identified through these motifs.
(5) Expression of ganp transcription
Northern blot analysis detected 7 kb mRNA, but it was a very weak signal compared to the control β-actin signal, and its expression was highly ubiquitous in all cell lines, organs and tissues tested. It was. In order to examine whether or not ganp mRNA is enhanced in the same region as that detected on the section by 29-15 mAb, in-situ RNA hybridization analysis was performed, and ganp mRNA was immunized with SRBC. Abundantly expressed in the central region of the splenic GC, while not expressed in the unimmunized spleen (FIG. 11), thymus, and lymph nodes. Although ganp mRNA was enhanced in GC-B cells of immunized mice, this expression pattern was very similar to the results with 29-15 mAb based on hematoxylin staining on the same section. In non-immune BALB / c mice, ganp mRNA was also enhanced in the GC region of PP, and the expression of ganp mRNA was prominent in the plasma-like cells in the spleen red pulp region of non-immune NZB mice. (FIG. 11). These results suggest that the ganp gene encodes a molecule that is recognized by 29-15 mAb.
(6) GANP expression in B cells
By anti-GANP mAb (42-23), a single 210 kD protein band was detected from both the nucleus and cytoplasmic compartments of WEHI-231 cells (FIG. 12). Next, in order to find evidence that GANP is functionally related to the activation and differentiation of B-lineage cells, non-immune BALB / c mouse B cells were stimulated in vitro using anti-IgM and anti-CD40 in combination. As a result, the expression of GANP protein detected by anti-GANP mAb increased (FIG. 13). Kinase reaction with in vitro GANP immunoprecipitates increased kinase activity bound to GANP protein in splenic B cells stimulated in vitro. Therefore, phosphorylation is induced in serine / threonine residues in GANP protein (FIG. 14). These results suggested that GANP may activate B cells in the peripheral immune response.
In addition, stimulation with anti-μAb and anti-CD40 mAb showed a maximal response, but either one showed only a slight response (data not shown). This activation was also detected by an increase in ganp mRNA in B cells stimulated by anti-μ and anti-CD40 co-ligation in vitro (FIG. 16). RT-PCR clearly demonstrated an increase in the amount of ganp mRNA at 24 and 48 hours post stimulation compared to control HPRT mRNA.
Since the 210 kDa GANP protein has a large number of potential phosphorylation sites, we tested the induction of phosphorylation by an in vitro kinase reaction with anti-GANP immunoprecipitates. As shown in FIG. 17, 210 kDa protein phosphorylation was seen in anti-GANP immunoprecipitates from splenic B cells stimulated by anti-μ and anti-CD40 co-ligation. This result indicates that the kinase activity is maintained even when GANP is precipitated.
(7) Association between GANP and MCM3 protein
We found a Map80 homology region (76.3% identity at the amino acid level) at the carboxyl terminus of GANP. Map80 is an 80 kDa nucleoprotein involved in the translocation between the cytoplasm and nucleus of MCM3 (protein essential for DNA replication) (Takei, Y. et al., 1998, J. Biol. Chem. 273: 22177-22180; Kimura, H. et al., 1994, EMBO J. 13: 4311-4320; Cong, JP et al., 1996, Trends Biochem. Sci. 21: 102-106; and Romanowski, P. et al., 1996, Curr. 6: 1416-1425). Therefore, we examined the interaction between GANP and MCM3 in WEHI-231. We detected that MCM3 was included in the anti-GANP immunoprecipitate. Since the phosphorylation state of MCM protein appears to be critical in regulating cell cycle progression (Kimura, H. et al., 1994, EMBO J. 13: 4311-4320; Chong, JP et al., 1996, Trends Biochem. Sci.21: 102-106; and Romanowski, P. et al., 1996, Curr. Biol.6: 1416-1425), anti-MCM3 immunoprecipitates were performed in vitro kinase assays. Due to MCM3 immunoprecipitation, a phosphorylated protein simultaneously precipitates at a position of 210 kDa, which is the same size as GANP (left panel in FIG. 19). These 210 kDa bands from anti-GANP and anti-MCM3 immunoprecipitates show the same pattern in V8 cleavage mapping (right panel of FIG. 19), indicating that GANP and MCM3 are associated in the B cell line. It is suggested.
Next, we tested whether MCM3 is activated in GC-B cells by antigen immunization of mice in vivo. Sections adjacent to those used above were stained with anti-MCM3Ab (FIG. 20). MCM3 is also activated in GCs. Double staining clearly demonstrated the co-localization of both MCM3 and PNA. A part of the GC region is strongly surrounded by FDCs (lymph follicular cells). These results indicate that MCM3 is a centroblast and GANP that is largely surrounded by FDCs. ⁺ It was demonstrated to be activated in GC-B cells containing centrosite (FIG. 20).
(8) Consideration
As described above, the present inventors have found GANP, which is a novel protein expressed in GC-B cells localized in the light region of the spleen secondary follicle. Under normal conditions, trace amounts of ganp mRNA can be detected in many types of cells, but GANP protein is enhanced in specific GC regions of immunized mice. Numerous studies have shown various differentiation antigens in GC as molecules recognized using mAbs or by specific cDNA cloning (Christoph et al., Int. Immunol., 6: 1203-1212). Li et al., Proc. Natl. Acad. Sci. USA., 93: 10222-10227, 1996; Kuo et al., J. Exp. Med., 186: 1547-1556, 1997). Most molecules appear in all regions of GC-B cells, but 8-oxoguanine DNA glycosylase is expressed in the dark region (Kuo et al., J. Exp. Med., 186: 1547-1556, 1997).
It is interesting that the GANP antigen is selective in the centrosite, which is the bright region. Recent studies have shown that the RAG protein required for immunoglobulin gene rearrangement is selectively expressed in the light region centrosite (Hikida et al., Science (Wash. DC), 274. 2092-2094, 1996; Han et al., Science (Wash. DC), 274: 2094-2097, 1996). As shown by Papabasiliou et al. And Han et al. (Papavasiliou et al., Science (Wash. DC), 278: 298-301, 1997; Han et al., Science (Wash. DC), 278: 301-305. , 1997), the GC region provides a site for secondary Ig gene rearrangement that occurs during T cell-dependent antibody responses, so GANP protein is responsible for maturation of antigen-specific B cells at the centrocytic stage. It may be a related component.
We have found that the carboxyl terminal portion of GANP has significant similarity to human Map80 (Takei, Y et al., 1998, J. Biol. Chem. 273: 22177-22180) which facilitates nuclear translocation of MCM3. . Immunoprecipitation experiments demonstrated that GANP also binds to MCM3 in WEHI-231. MCM3 is an MCM protein family essential for initiation of DNA replication (Kimura, H. et al., 1994, EMBO J. 13: 4311-4320; Blow, JJ 1993. J. Cell Biol. 122.993-1002. Tye, BK 1994. Trends Cell Biol.4: 160-166; Cong, JP et al., 1996, Trends Biochem.Sci.21: 102-106; Romanowski, P. et al., 1996, Curr. Biol.6: 1416-1425 and Thommes, P. et al., 1992, Nucl. Acids Res. 20: 1069-1074). The main fraction of nuclear MCM protein binds to chromatin at the start of S phase, but dissociates during replication and accumulates as free protein in the nucleus. Release of MCMs from chromatin is achieved by phosphorylation of multiple MCM proteins, and their reassociation after mitosis is accompanied by their dephosphorylation. MCM protein does not synthesize in differentiating cells with suppressed growth and appears to disappear at a rate related to half-life (Musahl, C., et al., 1998, Exp. Cell. Res. 241, 260- 264). The MCM3 protein has recently been shown to be an early target for apoptotic proteolysis (Schwab, BL et al., 1998, Exp. Cell Res. 238: 415-421). Schwab, B.M. L. Others propose that active disruption of MCM3 plays a role in inactivating the MCM complex and preventing out-of-date DNA replication during the execution of the cell death program. Our results indicate that GC-B cells express high levels of MCM3, some of which are associated with GANP. However, it is strange that proteins activated in differentiated cells that have halted the cell cycle bind to other proteins essential for S phase progression. It is speculated that the function of GANP is inactivation of the MCM3 protein through binding. The immunohistochemical data is consistent with the following idea. GANP is activated in centrosites that have stopped growing, whereas MCM3 is also expressed in rapidly cycling centroblasts and GCs. The amount of MCM3 will decrease by stopping gene expression and active disruption (Musahl, C., et al., 1998, Exp. Cell. Res. 241, 260-264; and Schwab, BL. Et al., 1998, Exp.Cell Res.238: 415-421), inactivation of MCM3 still expressed in centrosite through interaction with GANP may be another mechanism to prevent DNA replication. Furthermore, both GANP and MCM3 become phosphorylated by co-precipitating kinase (FIG. 19). Because highly phosphorylated MCM3 is thought to be an inactivated form (Kimura, H. et al., 1994, EMBO J. 13: 4311-4320), association with GANP may stimulate phosphorylation of MCM3 unknown.
Furthermore, the GANP protein has a close homology with the yeast Saccharomyces cerevisiae SAC3 molecule (SAC, actin suppressor), which is a suppressor of the temperature-sensitive mutation (act1-1) in the actin gene. It was isolated by genetic screening (FIG. 10; Novick et al., Genetics, 121: 659-674, 1989). The SAC3 protein is expressed in the nucleus and is required for protection against normal progression of mitosis and loss of chromosomes (Bauer et al., J. Cell Sci., 109: 1575-1583, 1996). Also, SAC3 null mutants grow very slowly and are larger than wild type cells. SAC3 is involved in processes that affect both the actin cytoskeleton and mitosis, indicating that SAC3 regulates gene expression of actin or actin-binding protein.
The gene that increases transcription of leucine permease activity in Saccharomyces (referred to as LEP-1) is identical to SAC3 (Stella et al., Yeast, 11: 460-460, 1995). The LEP-1 gene causes enhancement of yeast leucine permease, which is involved in selective amino acid transport, but is poorly understood for molecules involved in amino acid transport, particularly amino acid permeation in eukaryotic cells (Mastroberardino et al.,). Nature (Lond.), 395: 288-291, 1998). The SAC3 / LEP-1 sequence does not show a motif homologous to many transcription factors, but from already determined biological functions (Bauer et al., J. Cell Sci., 109: 1575-1583, 1996) It has been suggested that this sequence has regulatory activity of various target genes in the nucleus. Although mouse GANP does not display a typical consensus motif for nuclear transcription factors, it has a common ancestor with the yeast SAC3 gene, possible phosphorylation sites, two nuclear localization sequences, and other transcription molecules It is structurally similar to two supercoiled structures that can interact with.
GANP is selectively activated in Ag immunized splenic centrosites. It is also useful as a differentiation marker to identify centrocytic subsets that interact closely with FDCs in the GC region. Our studies have shown that BCR signals and CD40 costimulation together cause activation of GANP and induce signaling through the GANP / MCM3 complex.
The defective gene in autoimmune recessive genetic disease autoimmune polyglandular endocrine disorder (APECED) is identified by linkage analysis of human chromosome 21 (21q22.3), which is a possible transcriptional regulator. It encodes the AIRE gene product (Nagamine et al., Nature Genet., 17: 393-398, 1997) and this autoantibody recognizes the AIRE protein expressed in adrenal glands and other gonad producing tissues. APECED studies suggest that the involvement of molecules with nuclear co-activator activity may be related to autoimmunity. Both AIRE and GANP proteins do not have the typical domain of a transcriptional regulator, but have the LXXLL motif as observed in nuclear transcription co-activators as well.
B-cell specific nuclear co-activators (Bob1 / OCA-B / OBF1) have recently been characterized as cell type specific regulators Oct1 and Oct2 (Luo et al., Mol. Cell. Biol., 15: 4115-4124, 1995). In OCA-B target mice, after immunization with a T-dependent antigen, impaired GC formation is observed in the spleen, suggesting that B cell maturation is functionally involved in the GC region. (Kim et al., Nature (Lond.), 383: 542-547, 1996; Qin et al., EMBO J., 17: 5066-5075, 1998). GANP protein may be expressed under the control of OCA-B cells in centrosite, and the issue of molecular interaction of nuclear co-activator molecules is important for understanding B cell maturation in GC I think that the.
The New Zealand model of SLE is an experimental material for genome linkage studies to map the chromosomal location of disease susceptibility genes. Nephritis and autoantibody production on chromosome 4 (referred to as Nba1), on chromosome 7 and on chromosome 1 (referred to as Nba2; Vyse et al., J. Immunol., 158: 5566-5574, 1997) At least 12 non-MHC loci linked to each were mapped independently. GANP antigen on large cells is highly enhanced in the red pulp region of non-immunized NZB mice (FIGS. 4-8), but NZB mice have a similar large IgM called Mott cells in the red pulp region. Had production cells. Mott cells appear selectively in NZB and (NZB x NZW) F1 mice but not in normal BALB / c or C57BL / 6 mice.
Mott cell progenitor cells are probably B-1B cells (Tallinton et al., Eur. J. Immunol., 22: 531-539, 1992; Jiang et al., J. Immunol., 158: 992-997, 1997), this suggests that B cells are closely related to autoimmunity. Mott cells are identified by inclusion bodies of IgM in the cytoplasm and stained positively with PAS (Tallinton et al., Eur. J. Immunol., 22: 531-539, 1992; Jiang et al., J. Immunol. , 158: 992-997, 1997). GANP ^{dense +} Since the cells seemed to be Mott cells, PAS staining was performed, and GANP in the red pulp region of NZB mice was examined. ^{dense +} Cells are PAS ⁻ Met. GANP ^{dense +} IgM producing cells appear in the spleen of NZB mice as well as Mott cells, but these cells were different. Activation of an abnormal B cell population associated with one of the chromosomal loci linked to disease susceptibility may occur, but this activation may result in a new type of IgM producing cell.
Lyn reported by several research groups ^{− / −} Mouse and CD40L ^{− / −} Mice show similar autoimmunity and high IgM syndrome, but in these conditions there are many inclusions-containing immunoblasts in the spleen (Hibbs et al., Cell, 83: 301-311, 1995; Nishi et al. al., Immunity, 3: 549-560, 1995; Xu et al., Immunity, 1: 423-431, 1994). These observations suggest that the transduction of signals by BCR and CD40 regulates the production of abnormal antibody-producing plasma cells. In addition, stimulation of splenic B cells with anti-IgM and anti-CD40 antibodies induces GANP protein phosphorylation activity. From this observation, GANP protein may be related to the downstream of B cell activation site in the GC region. It was suggested that abnormal B cell activation in NZB mice may be related to increased GANP protein expression.
Example 2: Cloning of the human GANP gene
Based on the sequence information of the rat GANP gene, the human GANP gene was cloned and sequenced. Specifically, using λgt11-human heart cDNA library (Clontech), gspl-1: TTTGTCTGGAGGATGATCGC (SEQ ID NO: 7 in the sequence listing), gspl-2: AAAGAGAAAAGGGGCCAGGCC (SEQ ID NO: 8 in the sequence listing), and primers gspl-3: CCAGCTTCTTGTCCAAAAGC (SEQ ID NO: 9 in the sequence listing) was used, and further 5'RACE System for Rapid Amplification of cDNA Ends, Version 2.0 (GibcoBRL) was used for cloning and determination of the base sequence. It was.
The base sequence of the obtained clone was determined. The nucleotide sequence of the obtained human GANP gene is shown in SEQ ID NO: 4 in the sequence listing. The amino acid sequence encoded by this base sequence is shown in SEQ ID NO: 3 and FIG. The human GANP gene showed high homology with the mouse GANP gene, and human GANP contained an 80-kDa Map80 domain on the carboxyl-terminal side.
By in situ RNA hybridization, the ganp transcript appeared to be activated in the GC region of the tonsils. GANP ⁺ The cell is a memory B cell CD38 ⁺ IgD ⁻ Expresses phenotype. These results indicated that human GANP is also expressed in GC-B cells in secondary lymphoid tissues. Further, human GANP consisting of 1980 amino acids has a stretch of Map80 homology region that binds to MCM3 protein in B cells, suggesting that GANP is involved in cell cycle regulation in GC-B cells.
Furthermore, in situ hybridization was performed by the FISH method using the obtained human GANP gene and a human chromosome sample. The results are shown in FIG. As can be seen from FIG. 22, the genomic fragment having the human GANP gene and Map80 was mapped to chromosome 21 long arm 22.3.
Industrial applicability
The protein of the present invention is a novel protein having kinase activity and may be involved in signal conversion of abnormal B cell differentiation in an autoimmune state. Therefore, the protein, polypeptide, polynucleotide, antisense polynucleotide and antibody of the present invention are useful for elucidating the mechanism of autoimmunity.
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 shows 29-15 in PP of normal mice ⁺ It is the photograph which showed detection of the cell. Immunohistochemical analysis was performed on PP using 29-15 mAb and ALP anti-rat Ig antibody. Positive cells appeared in the central region with the vector blue ALP substrate, and in the small intestinal villi containing non-specific endogenous ALP activity, a strong signal was observed in the peripheral region. To perform two-color staining, sections were further stained with either biotin anti-B220 mAb or biotin anti-IgD mAb and developed with DAB and HRP-streptavidin.
FIG. 2 shows 29-15 in the GC region of SRBC immunized mice. ⁺ It is the photograph which showed the appearance of the cell. Normal BALB / c mice were injected with SRBC four times in 12 days, and spleen sections were stained with hematoxylin or examined by immunohistochemical staining similar to FIG. Compared after staining with 29-15 mAb, sections of normal and SRBC immunized BALB / c mice were similar.
FIG. 3 shows 29-15 in the GC region of SRBC immunized mice. ⁺ It is the photograph which showed the appearance of the cell. Sections of the GC area were stained with PNA, anti-BrdU and 29-15 mAb in combination with the individual colors described in “Materials and Methods”. The upper photo shows hematoxylin staining of the GC region (GC) and central groove artery (CA), the middle photo is 29-15. ⁺ PNA ⁺ Three color staining pointing to the cells is shown. The bottom is 29-15 ⁺ PNA ⁺ A cell is illustrated.
FIG. 4 shows GANP in the red pulp region of autoimmune-prone mice ^{dense +} It is the photograph which showed the expression of the cell. BALB / c, NOD, NZB, (NZB x NZW) F ₁ Sections were prepared from the spleens of non-immune mice of the strain, BXSB strain, and MRL / lpr strain. All mice were used at 6-8 weeks of age. GANP stained with 29-15 mAb ^{dense +} Cells are NZB, (NZB x NZW) F ₁ Appeared in the splenic spinal cord of MRL, MRL / lpr and BXSB strains.
FIG. 5 shows GANP in the red pulp region of autoimmune-prone mice ^{dense +} It is the photograph which showed the expression of the cell. LN sections of the popliteal area were stained with 29-15 mAb. GANP ^{dense +} Cells appeared in the peripheral LN of aged NZB mice (10 months old) and MRL / lpr mice (8 weeks old).
FIG. 6 shows GANP in autoimmune propensity mice. ^{dense +} It is the photograph which showed the characterization of the cell. Spleen sections of non-immunized NZB mice (8 weeks old) were prepared. Immunohistochemical analysis was performed using 29-15 mAb in combination with one of the following reagents: anti-B220, PNA.
FIG. 7 shows GANP in autoimmune propensity mice. ^{dense +} It is the photograph which showed the characterization of the cell. Spleen sections of non-immunized NZB mice (8 weeks old) were prepared. Immunohistochemical analysis was performed using 29-15 mAb in combination with one of the following reagents: anti-IgM, anti-Syndecan-1, anti-BrdU mAb.
FIG. 8 is a photograph showing Mott cells appearing in NZB mice by PAS staining.
FIG. 9 is a diagram showing the deduced amino acid sequence of mouse GANP protein with one letter mark.
FIG. 10 is a diagram showing the structure of the GANP protein. In the figure, S / T rich region: a region rich in serine / threonine, SAC3 homology region: SAC3 homologous region, and nuclear localization signal: nuclear localization signal. There are four LXXLL motifs.
FIG. 11 is a photograph showing the results of in situ RNA hybridization of the ganp gene. Spleen sections from SRBC immunized, non-immunized BALB / c and NZB mice were hybridized using a ganp antisense probe. In the figure, a white spleen spinal cord region (WP), a red splenic spinal cord region (RP), and a GC region (GC) are shown. GANP in the red pulp region of NZB mice ^{dense +} Cells were observed.
FIG. 12 shows the results of analyzing the GANP protein by Western blot after immunoprecipitation. GANP protein was detected as a 210 kD protein expressed in the cytoplasmic and nuclear fractions of WEHI-231 cells.
FIG. 13 shows splenic B cells of normal BALB / c mice, F (ab ′) of goat anti-IgM antibody (10 μg / ml) and anti-CD40 mAb (10 μg / ml). ₂ It is the figure which showed the result which stimulated for 48 hours and was dye | stained with anti- GANP mAb.
FIG. 14 shows anti-GANP immunoprecipitates using [γ- ³² It is the figure which showed the result of having implemented the in vitro kinase reaction for 10 minutes in presence of P] -ATP. Protein phosphorylation was detected by autoradiography after separation by SDS-PAGE. The phosphorylation of GANP is indicated by arrows (Fig. A), and phosphoamino acid analysis of phosphorylated GANP protein is also shown (Fig. B).
FIG. 15 shows the structure of mouse GANP protein. In the figure, regions homologous to SAC3 and Map80, nuclear localization sequences (NLSs), and coiled-coil regions are shown. Four LXXLL motifs are shown in black.
FIG. 16 shows the results of RT-PCR analysis. Activation of ganp mRNA in anti-μ- and anti-CD40-stimulated B cells in vitro has been shown. HPRT was used as a control to check the amount of each template.
FIG. 17 shows the results of the in vitro kinase reaction. Cell lysates were prepared from unstimulated (left) or stimulated (right) cells and subjected to anti-GANP immunoprecipitation. The in vitro kinase reaction is [γ- ³² P] was performed with anti-GANP (42-23) immunoprecipitate in the presence of ATP for 10 minutes. Protein phosphorylation was detected by autoradiography after SDS-PAGE separation. The arrow indicates the position of phosphorylated GANP.
FIG. 18 is a diagram showing a physical association between GANP and MCM3. Cell lysates from WEHI-231 were immunoprecipitated with anti-GST, anti-GANP (42-23), or anti-MCM3 Ab. After separation by SDS-PAGE, the protein was transferred to a membrane by electrophoresis, and anti-MCM3-Ab was detected as a probe.
FIG. 19 shows the physical association between GANP and MCM3. Anti-GST, anti-GANP (42-23) and anti-MCM3 immunoprecipitates from WEHI-231 cell lysates were subjected to in vitro kinase assays. Normal rabbit serum (NRS) was used as a control for anti-MCM3 Ab. Samples were separated by 7% SDS-PAGE. Bands corresponding to GANP and MCM3 are indicated by arrows in the left panel. In the right panel, the V8 cleavage mapping of the 210-kDa band showed the same cleavage pattern. As a control, an irrelevant V8 digested protein was separated in parallel.
FIG. 20 shows the results of double staining of anti-MCM3 Ab and anti-CR1 mAb or PNA. MCM3 expression was activated in the GC region.
FIG. 21 is a diagram showing the deduced amino acid sequence of human GANP protein in one-letter code.
FIG. 22 is a photograph showing the result of mapping human ganp and Map80 by the FISH method using human chromosomes.

Claims (5)

  A GANP protein specified by the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3 in the sequence listing.   In the amino acid sequence described in SEQ ID NO: 1 or 3 in the sequence listing, one or more amino acids are deleted, one or more amino acids are substituted with other amino acids, and / or one or more other A GANP mutant protein comprising an amino acid sequence to which is added an amino acid and having kinase activity.   A polynucleotide encoding the protein according to claim 1 or 2.   The antisense polynucleotide which consists of a base sequence of the antisense strand of the polynucleotide of Claim 3.   An antibody that recognizes the protein according to claim 1 or 2.

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